Human and animal data demonstrate that cholesterol (CLR) may disrupt the smooth muscle (SM) tone of resistance-size cerebral arteries in absence of any sign of arteriosclerosis or anatomical abnormalities, this CLR action contributing to a vascular component of stroke. In a further step, stroke patients with dysfunction of large cerebral arteries and high CLR will present a higher prevalence of arteriosclerosis. CLR actions on cerebral artery SM tone have been attributed to endothelial, microcirculation, inflammatory and other circulating factors. Departing from this current paradigm and supported by preliminary data, our central hypothesis is that CLR may control resistance-size, cerebral artery SM tone and diameter via regulation of calcium- and voltage-gated potassium channels of big conductance (BK) located in the cerebral artery SM plasmalemma. Here, BK function primarily results from the coupling of channel-forming ? (cbv1) and regulatory ?1 subunits. The concerted action of cbv1+?1 and eventual BK activation generates outward potassium current that opposes depolarization- mediated calcium influx, limits SM contraction and favors artery dilation. Based on preliminary data, we predict that under low ?1 expression, CLR interaction (through direct binding and allosteric modulation) with selected Cholesterol Recognition Amino acid Consensus motifs (CRACs) identified in the cbv1 cytosolic domain will lead to CLR-induced reduction of BK current, SM contraction and cerebral artery constriction. In contrast, under high ?1 levels, CLR-driven increases in the plasmalemmal fraction of ?1 and cbv1-?1 functional coupling will prevail, leading to increased BK current, SM relaxation and artery dilation. We predict that brain arteries that differ in ?1 expression will display a differential vulnerability to CLR, and that CLR levels in SM will condition the efficacy of ?1-dependent vasodilators. Our predictions will be tested along three specific aims (SA). SA1 (molecular level): determine the structural bases and gating mechanisms that lead to CLR-induced hindering of BK (cbv1 homotetramer) function through CLR-cbv1 CRAC interactions. SA2 (cellular level): determine the mechanisms that underlie CLR activation of cbv1+?1 channels. Knowledge from SA1 and SA2 will be integrated in SA3 (organ level): determine the consequences of CLR-BK subunit interactions on the function of native BKs in cerebral artery SM and on artery diameter under physiological conditions using in vitro and in vivo CLR delivery methods. We combine unique expertise in computational modeling, binding assays, mass spectroscopy, differential scanning fluorimetry, patch-clamp and lipid bilayer electrophysiology, allosteric gating analysis, electroporation of SM cells and cerebral arteries from engineered mice with mutated cDNAs, subunit trafficking, confocal imaging and cerebral artery diameter determinations, ensuring feasibility. We expect to challenge the paradigm that CLR modulation of BK is secondary to nonspecific perturbation of bilayer physico-chemical properties, and to provide milestone information on CLR control of cerebral artery function, which will be necessary to design small drugs that adjust BK channel function to variable CLR levels and counteract CLR-associated cerebrovascular disease.